1) Molecular machinery regulating protein secretion in the acinar cells of salivary glands [unreadable] The major secretory units in the salivary glands are the acini that are formed by pyramidal polarized cells, which form a small lumen where salivary proteins and water are secreted. Proteins destined to secretion are packed in secretory granules in the trans-Golgi network (TGN) from where they are transported throughout the cytoplasm to the cell periphery. Here, upon stimulation of either muscarinic or adrenergic receptors, the granules fuse with the apical domain of the plasma membrane (PM) releasing their content into the apical lumen. [unreadable] Our aim is to study the molecular machinery regulating the formation of the granules at the TGN and their fusion with the PM. Due the lack of reliable cell culture models, key components of this machinery in the salivary glands are still largely unknown, while in other exocrine glands, like the pancreas, substantial progress has been made. [unreadable] We plan to study these mechanisms by using three different and complementary model systems and namely, live animals, three-dimensional (3D) cell culture systems and organ explants.[unreadable] a) Establishing an animal model to study regulated protein secretion in mice[unreadable] To study protein secretion in live animals by intra-vital multi-photon microscopy, we are planning to engineer mice to express fluorescently labeled proteins that localize to the secretory granules. Our aim is to follow in real time the agonist-stimulated release of the granules in control conditions or when specific genes of interest have been ablated via viral mediated expression of short hairpin RNA (shRNA). As a first step, we selected the proteins to be expressed as transgenes, fused them with various fluorescent tags, and expressed them in cultured cells to check whether the addition of the tag could affect their expression, localization and function. We are now in the process of generating various transgenic animal lines expressing the selected genes.[unreadable] b) Establishing a three-dimensional cell culture system to study the mechanisms of protein secretion[unreadable] Cells derived from the intercalated ducts of human sub-mandibular glands (HSG) are believed to differentiate into acini, ducts, or myoepithelial cells depending on the growth conditions. While HSG cells grown on plastic or glass do not express any of the secretory markers characteristic of the differentiated salivary glands cells and do not show any polarization, remarkably, when grown on basement membranes they assume an acinar phenotype and express secretory proteins. However, it was not established whether these cells are competent to secrete cargo proteins and whether they have established polarized domains. We have shown that these 3D-grown HSG cells are not fully differentiated since they express both myoepithelial and acinar markers and furthermore, they are only partially polarized since only some molecules are properly targeted to the correct domain of the plasma membrane. We have also observed that growing the cells on a substrate mimicking the basal membrane promotes the activation of the sorting machinery at the TGN but the cells are not still competent to secrete. These finding suggest that key components other than the basal membranes are necessary for the complete differentiation of the HSG cells into fully functional secretory units and we are currently investigating the nature of these factors. [unreadable] [unreadable] 2) Molecular machinery regulating the sorting of proteins from the trans-Golgi network to the apical or the basolateral domain of the plasma membrane [unreadable] Proteins are sorted from the TGN to the apical or the basolateral domain of the PM. Whether these proteins follow a direct route to the PM or they are first diverted towards other intracellular compartments is still a matter of debate. Furthermore, the molecular machineries regulating the sorting events are not still understood. We are focusing our attention on two proteins belonging to a family of water channels expressed in acinar cells, aquaporin 3 (AQP3) and aquaporin 5 (AQP5), which are localized to the basolateral and the apical domain respectively. Our strategy is to express in live animals fluorescently tagged AQP3 and AQP5 by using viral-based expression vectors and to assess by time lapse imaging the route followed by these proteins once they are released from the TGN. Furthermore, we are introducing some mutations in the two proteins in order to establish the key determinant(s) regulating their sorting.[unreadable] [unreadable] 3) Molecular machinery regulating endocytosis in salivary glands of live animals [unreadable] The role of endocytosis in the physiology of the salivary glands has never been elucidated. Uptake of proteins from both the apical and the basolateral domains of the ductal system have been described but never thoroughly investigated. The presence under physiological conditions of salivary proteins in the bloodstream and of serum proteins in the saliva, argues strongly in favor of a constant and bi-directional transcytotic movement of proteins across the salivary gland epithelium. The salivary glands offer a unique opportunity to study endocytosis in polarized epithelial cells in a physiological context since both the basolateral and the apical domains are accessible from the bloodstream and from the major excretory duct, respectively. [unreadable] Our aim is to define the endocytic pathways in the salivary glands, to investigate the molecular machinery regulating the internalization of various cargo with a particular emphasis on the role of the cytoskeleton, and finally, to understand what is the contribution of the endocytic events in the physiology of the glands and especially during secretion. [unreadable] Characterization of the endocytic pathways in rat submandiular glands of live rodents[unreadable] To study the endocytosis in live rodents we injected systemically fluorescent probes, like dextrans and transferrin, that in cell cultures are internalized by different mechanisms and we imaged their uptake in the parenchyma of the submandibular salivary glands by using intra-vital multi-photon microscopy. We found that both acinar and myoepithelial cells internalize effectively the various probes from the basolateral domain while the ductal cells showed a limited internalization. We compared the endocytosis in salivary glands with that of other organs like the kidneys, the liver and the spleen, which show a much higher rate of internalization, most likely reflecting distinct permeability to the probes and a different degree of vascularization. We are currently investigating further these mechanisms and we are in the process of engineering mice to express fluorescently labeled markers of the endocytic pathways (i.e. clathrin, caveolin and actin). Furthermore, we are preparing viral-based vectors to express various shRNA to screen for a series of genes that might be involved in the early steps of internalization.